Chimeric HIV fusion proteins as vaccines

ABSTRACT

A method for inducing HIV antigen-specific immune responses is disclosed. The method comprises administering to a subject in need thereof a therapeutically effective amount of a chimeric fusion protein comprising: (a) a first polypeptidyl region comprising a  Pseudomonas Exotoxin  A (PE) binding domain and a PE translocation domain, located at the N-terminus of the fusion protein; and (b) a second polypeptidyl region with a fusion peptide of HIV gp120-C1-C5-gp41 with the amino acid sequence of SEQ ID NO: 7. A method for inducing neutralizing antibodies against HIV-1 is also disclosed.

REFERENCE TO RELATED APPLICATION

This application is a divisional application of U.S. Ser. No.12/358,659, filed Jan. 23, 2009, which status is pending and claims thepriority of U.S. provisional application No. 61/025,094, filed Jan. 31,2008, all of which are herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

The present invention relates generally to HIV vaccines, and morespecifically to chimeric HIV fusion proteins useful for inducing humoraland cell-mediated immune responses.

BACKGROUND OF THE INVENTION

The global epidemic of AIDS has created an urgent need for a vaccineagainst human immunodeficiency virus type 1 (HIV-1). It is likely thateffective AIDS vaccines will need to generate efficient humoral andcellular immune responses. Virus-neutralizing antibodies and anti-HIVcytotoxic (CD8+) T lymphocytes (CTLs) mediated immunity are majorrequirements for protective immune responses elicited by HIV vaccines.

HIV has several major genes coding for viral proteins. The gag genecodes for p24, the viral capsid; p6 and p7, the nucleocapsid proteins;and p17, a matrix protein. The pol gene codes for reverse transcriptase,integrase, and protease which cleaves the proteins derived from gag andpol into functional proteins. The env gene codes for the precursor togp120 and gp41, envelope proteins embedded in the viral envelope thatenable the virus to attach to and fuse with target cells. The tat, rev,nef, vif, vpr, vpu genes each codes for a single protein with the samenames, Tat, Rev, Nef, Vif, Vpr, Vpu, respectively.

Neutralizing antibodies have been shown to contribute to protection fromvirus infection in animal models of HIV-1 infection. The virus-specifictargets on HIV-1 accessible to neutralizing antibodies are the envelopeglycoproteins (Yang, X. et al. (2005) “Stoichiometry of AntibodyNeutralization of Human Immunodeficiency Virus Type 1” Journal ofVirology 79: 3500-3508). During the normal course of HIV-1 infections,virus-neutralizing antibodies are often generated but the titer ofneutralizing is often low. Most neutralizing antibodies bind the gp120envelope glycoprotein, which is the major exposed protein of the viralenvelope glycoprotein trimer. The more conserved receptor-bindingsurfaces of the HIV-1 gp120 glycoprotein are also the targets forneutralizing antibodies. The CD4-binding site (CD4BS) antibodiesrecognize a conformational epitope composed of several segments of gp120region that overlaps the binding site for CD4. CD4-induced (CD4i)antibodies bind a highly conserved gp120 element that is critical forthe gp120-chemokine receptor interaction. The ability of CDBS and CD4iantibodies to interfere with receptor binding contributes to theirneutralizing capability.

GP 120 contains ten domains: conserved domains 1-5 (C1-C5) and variabledomains 1-5 (V1-V5). The C1 and C5 domains are located at N- andC-terminals of gp120, respectively. Antibodies directed against the V3loop, which determines chemokine receptor choice, can block the bindingof gp 120 to the receptors CCR5 and/or CXCR4. Neutralization by anti-V3antibodies, although potent, is often limited to a small number of HIV-1strains.

Gp120 is non-covalently associated with gp41. The gp41 subunit isanchored in the membrane and has a non-polar fusion peptide at itsN-terminus. The gp120-gp41 complex forms oligomers on the surface ofinfected cells and on virions. The binding of gp 120 to CD4 is thoughtto result in activation of the membrane fusion activity of gp41, leadingto entry of the viral nucleocapsid into a cella Antibodies to gp41epitopes in the serum of HIV-infected individuals may play an importantrole in virus neutralization. Gp120-41 complex sequences of differentHIV subtypes show a remarkably conserved N-terminal coiled-coilstructures of gp41 as well as the C-terminal residues that interact withthe N-terminal core structure of gp120.

Multiple immune effectors participate in prevention, containment andclearance of HIV infection. To prevent infection of host target cells,antibodies are required. After the first target cells have been infectedwith virus, it is important to have cytotoxic T lymphocytes (CTLs) aswell as antibodies to reduce cell-to-cell spread and kill infectedcells. An effective HIV vaccine should evoke antibodies that can bind tovirus and prevent attachment of virus to target cells, as well as CTLsthat can eliminate any cells that become infected.

It remains a difficult goal for vaccinologists to constructlive-attenuated viruses that are both effective and safe, or to mimicthe presentation of viral proteins observed in infection withrecombinant antigens or with replicating or non-replicating vectorscarrying appropriate genes or antigens. The large number of mutations inthe V3 domain of gp120 has limited its usefulness as a target for HIVvaccine. It is still unclear how the trend of hypervariability in thevariable domains is developing and how many domains are absolutelyinvariant in the evolving strains of HIV.

A previously unaddressed need exists in the art to address thedeficiencies and inadequacies in HIV vaccine antigen production,especially in connection with the provision of efficacious, antigenicdeterminant peptides.

SUMMARY OF THE INVENTION

The importance of interaction between gp120 and gp41 for determinationof the neutralization phenotype has been studied. One aspect of theinvention relates to a chimeric fusion protein useful as an immunogenfor inducing HIV antigen-specific immune responses. The chimericcontains: (a) a first polypeptidyl region containing a PseudomonasExotoxin A (PE) binding domain and a PE translocation domain, located atthe N-terminus of the fusion protein; and (b) a second polypeptidylregion located at the C-terminus of the fusion protein, including: (i) afirst peptidyl segment containing a fragment of gp120 C1 domain, locatedat the N-terminus of the second polypeptidyl region; (ii) a secondpeptidyl segment containing a fragment of gp120 C5 domain, located atthe C-terminus of the first peptidyl segment; and (iii) a third peptidylsegment containing a fragment of gp41 amino acid sequence, located atthe C-terminus of the second peptidyl segment, wherein the secondpolypeptidyl region contains an antigenic determinant which is specificto one subtype of HIV. The one subtype of HIV is at least one selectedfrom the group consisting of HIV subtypes A, B, C, D, E, F, G, H, J andK.

In one embodiment of the invention, the fusion protein further includesan endoplasmic reticulum retention sequence, e.g., the amino acidsequence KDEL, at the C-terminus. In another embodiment of theinvention, the chimeric fusion protein further includes an intermediatepolypeptidyl region between the first and the second polypeptidylregions, in which the intermediate polypeptidyl region contains anon-Env, HIV antigenic determinant. In one embodiment of the invention,the intermediate polypeptidyl region is at least one selected from thegroup consisting of Gag24, Nef, Tat and Rev. In another embodiment ofthe invention, the intermediate polypeptidyl region includes Gag24 aminoacid sequence or a fragment thereof. Further in another embodiment ofthe invention, the intermediate polypeptidyl region contains an N- orC-terminus of Gag24 amino acid sequence. In one embodiment of theinvention, the intermediate polypeptidyl region contains the amino acidsequence set forth by SEQ ID NO: 151.

Another aspect of the invention relates to a chimeric HIV fusion proteinuseful as an immunogen for inducing HIV antigen-specific immuneresponses, which includes: (a) a first polypeptidyl region containing aPE binding domain and a PE translocation domain, located at theN-terminus of the fusion protein; and (b) a second polypeptidyl regioncontaining an HIV protein or a fragment thereof, located at theC-terminus of the fusion protein, wherein the second polypeptidyl regioncontains an antigenic determinant which is specific to one subtype ofHIV.

In one embodiment of the invention, the second polypeptidyl regioncontains a fragment of HIV Env. In another embodiment of the invention,the second polypeptidyl region contains one or more than one fragment ofgp120 V3 domain. In another embodiment of the invention, the secondpolypeptidyl region contains the amino acid sequence set forth by SEQ IDNO: 6. In one embodiment of the invention, the second polypeptidylregion comprises an HIV protein or a fragment thereof, which is selectedfrom the group consisting of Gag24, Nef, Tat and Rev.

Further in another embodiment of the invention, the second polypeptidylregion contains HIV Gag24 amino acid sequence or a fragment thereof.Further in another embodiment of the invention, the second polypeptidylregion is a chimeric protein that includes: (i) a first peptidyl segmentcontaining a fragment of gp120 C1 domain, located at the N-terminus ofthe second polypeptidyl region; (ii) a second peptidyl segmentcontaining a fragment of gp120 C5 domain, located at the C-terminus ofthe first peptidyl segment; and (iii) a third peptidyl segmentcontaining a fragment of gp41 amino acid sequence, located at theC-terminus of the second peptidyl segment. Yet in another embodiment ofthe invention, the second polypeptidyl region comprises the amino acidsequence set forth by SEQ ID NO: 7.

Yet another aspect of the invention relates to a method of inducing anHIV-antigen specific immune response. The method includes the step ofadministering an effective amount of the chimeric fusion protein asdescribed above in a biocompatible carrier fluid suitable for carryingand delivering a predetermined aliquot of the fusion protein to apre-chosen site in a living subject.

These and other aspects will become apparent from the followingdescription of the preferred embodiment taken in conjunction with thefollowing drawings, although variations and modifications therein may beaffected without departing from the spirit and scope of the novelconcepts of the disclosure.

The accompanying drawings illustrate one or more embodiments of theinvention and, together with the written description, serve to explainthe principles of the invention. Wherever possible, the same referencenumbers are used throughout the drawings to refer to the same or likeelements of an embodiment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1F are the maps of plasmids encoding chimeric HIV fusionproteins.

FIG. 2A is a graph showing ELISA titers in the sera samples from animalsimmunized with various chimeric HIV envelope fusion proteins. Serasample 1:2,500 dilution.

FIG. 2B is a graph showing ELISA titers in the sera samples from animalsimmunized with various chimeric HIV envelope fusion proteins. Serasample 1:12,500 dilution.

FIG. 3 is a graph showing neutralizing antibodies against live viruseswere induced in the mice immunized with HIV Env fusion protein vaccines.

FIG. 4 is a flow chart illustrating the construction of pPE(ΔIII)-HIVgag24-K3.

FIG. 5 is a flow chart illustrating the construction of pPE(ΔIII)-HIVgag24-gp120-41-K3.

FIG. 6 is a schematic drawing illustrating the fusion of an HIVantigenic determinant peptide to gp 120 C1-C5-gp41 creates a chimericthat enhances the antigenic determinant peptide's cell-mediated immuneresponse.

FIG. 7 is a flow chart illustrating the construction of pPE(ΔIII)-HIVnef-NC-K3.

DETAILED DESCRIPTION OF THE INVENTION Definitions

The terms used in this specification generally have their ordinarymeanings in the art, within the context of the invention, and in thespecific context where each term is used. Certain terms that are used todescribe the invention are discussed below, or elsewhere in thespecification, to provide additional guidance to the practitionerregarding the description of the invention. For convenience, certainterms may be highlighted, for example using italics and/or quotationmarks. The use of highlighting has no influence on the scope and meaningof a term; the scope and meaning of a term is the same, in the samecontext, whether or not it is highlighted. It will be appreciated thatsame thing can be said in more than one way. Consequently, alternativelanguage and synonyms may be used for any one or more of the termsdiscussed herein, nor is any special significance to be placed uponwhether or not a term is elaborated or discussed herein. Synonyms forcertain terms are provided. A recital of one or more synonyms does notexclude the use of other synonyms. The use of examples anywhere in thisspecification including examples of any terms discussed herein isillustrative only, and in no way limits the scope and meaning of theinvention or of any exemplified term. Likewise, the invention is notlimited to various embodiments given in this specification.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention pertains. In the case of conflict, thepresent document, including definitions will control.

As used herein, “around”, “about” or “approximately” shall generallymean within 20 percent, preferably within 10 percent, and morepreferably within 5 percent of a given value or range. Numericalquantities given herein are approximate, meaning that the term “around”,“about” or “approximately” can be inferred if not expressly stated.

As used herein, the term “carboxyl terminal moiety which permitsretention of the fusion antigen to the endoplasmic reticulum (ER)membrane of a target cell” refers to a peptide fragment that enables thefusion antigen to bind to the ER membrane and to retain it in the ERlumen for glycosylation and make it appears to be more like foreignprotein. In one embodiment of the invention, the carboxyl terminalmoiety comprises, in a direction from the amino terminus to the carboxylterminus, the following amino acid residues:R¹-R²-R³-R⁴-(R⁵)_(n)

Wherein,

R¹ is a positively charged amino acid residue;

R² is a negatively charged amino acid residue;

R³ is a negatively charged amino acid residue;

R⁴ is L;

R⁵ is a positively charged amino acid residue; and

n is 0 or 1.

Preferably, the carboxyl terminal moiety is a member of the KDEL familyprotein. As used herein, the term “KDEL family protein” refers to agroup of proteins, which has a similar carboxyl end binding to the ERmembrane of a cell and further has an ability for retention of suchprotein in the ER lumen. Generally, the length of the carboxyl endranges from 4 to 16 residues. As discussed in U.S. Pat. No. 5,705,163(which is incorporated herein by reference in its entirety), the aminoresidues at the carboxyl end of a KDEL family protein, particularlythose in the last five amino acids, are important. As shown in thestudies on the similar sequences present in different molecules andperforming a specific biological function, a sequence that retains anewly formed protein within the endoplasmic reticulum is Lys Asp Glu Leu(KDEL). These findings suggest that the sequence at the carboxyl end ofthe fusion antigen according to the invention acts as some type ofrecognition sequence to assist translocation of the fusion antigen froman endocytic compartment into the ER and retains it in the lumen. Thecarboxyl terminal moiety comprises the sequence of KDEL. For example,the carboxyl terminal moiety may comprise the sequence of KKDLRDELKDEL(SEQ ID NO: 250), KKDELRDELKDEL (SEQ ID NO: 251), KKDELRVELKDEL (SEQ IDNO: 252), or KKDELRXELKDEL, in which R is D or V.

The terms “PE(ΔIII)-HIV gp120” and “PE(ΔIII)-HIV gp120 V3-V3” areinterchangeable.

The terms “PE(ΔIII)-HIV gp120-41” and “PE(ΔIII)-HIV gp 120 C1-C5-gp41”are interchangeable.

The terms “HIV subtype A gp120 C1-C5-gp41” and “chimera A” areinterchangeable; the terms “HIV subtype B gp120 C1-C5-gp41” and “chimeraB” are interchangeable; the terms “HIV subtype C gp120 C1-C5-gp41” and“chimera C” are interchangeable, and so on.

Immunogens. To be an immunogen, the formulation need only be a mixtureof a fusion protein construct as described herein and a biocompatiblecarrier fluid suitable for carrying and delivering a predeterminedaliquot of the fusion protein construct to a prechosen site in the bodyof a living subject. Immunogens embodying the invention can beadministered in any appropriate carrier for intradermal, subcutaneous,intramuscular, parenteral, intranasal, intravaginal, intrarectal, oralor intragastric administration. They can be introduced by any means thateffect antigenicity in humans. The dosage administered will vary and bedependent upon the age, health, and weight of the recipient; the kind ofconcurrent treatment, if any; the frequency of treatment; and the natureof the humoral antibody response desired. If the immunogens are to begiven intradermally, subcutaneously, intramuscularly, intravenously orparenterally, they will be prepared in sterile form; in multiple orsingle dose formats; and dispersed in a fluid carrier such as sterilephysiological saline or 5% dextrose solutions commonly used withinjectables. In addition, other methods of administration can beadvantageously employed as well.

Vaccines. To be a prepared vaccine, the minimal formulation comprises apredetermined quantity of a fusion protein construct as describedherein; a biocompatible carrier suitable for carrying and delivering apredetermined aliquot of a fusion protein construct to a prechosen sitein the body of a living subject; and at least one adjuvant compositiondispersed in the carrier fluid or coupled to the fusion proteinconstruct. The vaccine, by definition, incorporates an immunogen andincludes one or more adjuvants to facilitate or stimulate the immuneresponse and to prolong the antigenic effect in-vivo over time. Amongthe useful adjuvant substances conventionally known are thosecompositions approved by the FDA (currently or pending for systemicand/or mucosal immunizations). Some are preferred formucosally-administered vaccines and others are preferred forintragastric administered vaccines.

Modes of administration. Multiple modes of inoculation, the manner ofintroducing an immunogen or vaccine, are conventionally known and used.The systemic or parenteral forms of administration (introduction byinjection or perfusion) typically include intraperitoneal, intravenous,intramuscular, subcutaneous, and subdermal inoculations. In contrast,mucosal modes of administration may include not only the intranasal andintragastric forms of introduction, but also oral, intravaginal, andintrarectal introductions.

EXAMPLES

Without intent to limit the scope of the invention, exemplaryinstruments, apparatus, methods and their related results according tothe embodiments of the present invention are given below. Note thattitles or subtitles may be used in the examples for convenience of areader, which in no way should limit the scope of the invention.Moreover, certain theories are proposed and disclosed herein; however,in no way they, whether they are right or wrong, should limit the scopeof the invention so long as the invention is practiced according to theinvention without regard for any particular theory or scheme of action.

I. HIV-1 gp120 and gp41 Fusion Proteins Example 1 Selection of TruncatedSegments from HIV Env Proteins, gp120 and gp41

The amino acid sequences of HIV gp120 and gp41 were retrieved from theNational Center of Biotechnology Information (NCBI, USA) database andentered into software for evaluation of antigenic determinant (epitopes)of the target proteins, and candidate segments for synthesis displayedon an evaluation plot. Antigenic determinant regions of the targetprotein were chosen for synthesis by a reverse genetic engineeringtechnique. Several peptide segments were selected as target peptidesbased on the results of the evaluation software. The software DNAstrider v1.0 was used to analyze whether the nucleotide sequences of thetarget peptides contained restriction enzyme sites. If present in theDNA sequence in disadvantageous places, changes were made within theappropriate codons without altering the amino acid sequence. Thesoftware checked the newly created sequence, and designed restrictionsites at both termini of the DNA sequence to facilitate cloning. Codonsfor some amino acid residues, such as Arg, Ile, Gln, Pro, were modifiedto increase the expression of proteins in E. coli expression systems.Table 1 lists the selected peptide segments and their correspondingamino acid sequences.

Example 2 Construction of Chimeric Target Polypeptides gp120 V3-V3 andgp120 C1-C5-gp41

Two target peptides gp 120 V3-V3 and gp120 C1-C5-gp41 were constructedusing the selected peptide segments as follows: Three truncated peptidesegments having the amino acid sequences of SEQ ID NO. 1 (from gp120 C1domain), SEQ ID NO. 2 (from gp120 C5 domain) and SEQ ID NO. 5 (from gp41region associated with gp120), respectively, were ligated to form achimeric target peptide gp120 C1-C5-gp41 (referred to as gp120-41). Twotruncated segments having amino acid sequences of SEQ ID NOs: 3 and 4(both from gp 120 V3 domains), respectively, were fused to formpolypeptide gp 120 V3-V3 (referred to as gp120). One or more residuesmight be inserted in-between to link two peptide segments. The number ofresidues inserted in-between was about 1 to 15 amino acids, which mightbe selected from amino acid residues that would not alter the secondarystructure of proteins, such as glycine, alanine, valine, and leucine.The amino acid residue cysteine in SEQ ID NO: 2 and in SEQ ID NO: 5could form a disulfide bond so that the chimeric target peptidegenerated could possess a three-dimensional structure.

TABLE 1 HIV Env Target peptide SEQ ID Proteins segmentsSequence of selected peptide segments No.  gp120 C1 domainVEKLWVTVYYGVPVWK 1 C5 domain KVVKIEPLGVAPTKCKRRVVQREKR 2 V3 domainCTRPSNNTRKGIHMGPGGAFYTTGQIIRNIRQAHC 3 V3 domainCTRPNNNTRRSIHIEPEGAFYTTGEIIGDIRQAHC 4 gp4l gp 41 region inQARVIAVERYLKDQQLLGIWGGSGKLICCTTAVP 5 association WNSSWSNKLDRIWNNMTWLEwith gp120

TABLE 2 Chimeric target SEQ ID peptide Amino acid sequence No. gp120 IGCTRPSNNTRKGIHMGPGGAFYTTGQIIRNIRQAHC GLLGGC 6 V3-V3

GLGLE gp120 VEKLWVTVYYGVPVWKKVVKIEPLGVAPTKCKRRVVQREKR 7 C1-C5-gp41GGGGGQ

LE

Table 2 lists the amino acid sequences of the chimeric target peptidesgp120 V3-V3 and gp120 C1-C5-gp41. For chimeric peptide gp120 V3-V3 intable 2: the underlined letters denote the restriction sites; boldletters denote the first V3 domain segment, bold and italic lettersdenote the second V3 domain segment. For chimeric peptide gp 120C1-C5-gp41 in table 2: the bold letters denote the C1 domain segment;non-bold letters denote the C5 domain segment; non-bold and italicletters denote linkers; bold and italic letters denote the gp41 domain;and the underlined letters denote a restriction site.

The chimeric target peptide gp120 V3-V3 (SEQ ID NO: 6) was designedbased on the construction of repeats in the V3 domain of gp120, and thechimera gp120 C1-C5-gp41 (SEQ ID NO: 7) was based on simulation of thegp41 region in association with gp120. The chimera gp120 V3-V3 (SEQ IDNO: 6) comprises amino acid sequences of SEQ ID NOs: 3 and 4 (both fromgp120 V3 domains). The chimera gp120 C1-C5-gp41 comprises amino acidsequences of SEQ ID NOs: 1 (from gp120 C1 domain), 2 (from gp120 C5domain), and 5 (gp41region in junction with gp120) (Table 1). Disulfidebonds were formed due to cysteines in SEQ ID NO: 2 (from the C5 domainof gp120) and SEQ ID No. 5 (from gp41 region in junction with gp120).

Using the similar method as described above, the following chimerictarget peptides were constructed: HIV subtype A gp120 C1-C5-gp41; HIVsubtype B gp120 C1-C5-gp41; HIV subtype C gp120 C1-C5-gp41; HIV subtypeD gp120 C1-C5-gp41; HIV subtype E gp120 C1-C5-gp41; HIV subtype F gp120C1-C5-gp41; HIV subtype G gp120 C1-C5-gp41; HIV subtype H gp120C1-C5-gp41; HIV subtype J gp 120 C1-C5-gp41; and HIV subtype K gp 120C1-C5-gp41 (abbreviated as chimeric target peptides A, B, C, D, E, F, G,H, J, and K, respectively).

These chimeras A, B, C, D, E, F, G, H, J and K were each constructedfrom a combination of 3 peptide segments selected from eachcorresponding HIV-1 subtypes, A, B, C, D, F, G, H, J and K,respectively. The basic scheme of the construction was to link twopeptide segments, each selected from the C1 and C5 domains of gp120, toanother segment selected from the gp41 region in association with gp120for each HIV-1 subtype. Thus, like the SEQ ID NO: 7, all these chimeratarget peptides have gp120-C1-C5-gp41-like structures.

Table 3 lists the amino acid sequences of these chimeras, in which thenon-bold, italic letters denote a segment from the C1 domain, the boldletters denote a segment from the C5 domain, of HIV subtype A gp120protein; the non-bold, non-italic letters GGGGG denote a linker, and thebold, italic letters denote a segment from HIV subtype A gp41 region injunction with gp120.

TABLE 3 Chimera for HIV SEQ ID subtype Amino Acid Sequence NO. AAENLWVTVYYGVPIWKKVVKIE PLGVAPTKARRRVVEREKRGGGGG 

31

B TEKLWVTVYYGVPVWKKVVKIE PLGIAPTKAKRRVVQREKRGGGGG 32

C MGNLWVTVYYGVPVWKKYKVVEIK PLGVAPTKPKRRVVEREKRGGG 33 GG 

D ADNLWVTVYYGVPVWKKVVQIE PLGVAPTRAKRRVVEREKRGGGGG 34

E SXNLWVTVYYGVPVWRKVVQIE PLGIAPTRPKRRVVEREKRGGGGG 

35

F ADNLWVTVYYGVPVWKKVVEIE PLGVAPTKAKRQVVQREKRGGGGG 36

G ASNNLWVTVYYGVPVWEDAKKVVKIK PLGVAPTKARRRVVGREKRG 37 GGGG 

H VVGNLWVTVYYGVPVWKKVVKIE PLGVAPTEARRRVVEREKRGGGG 38 G 

J AKEDLWVTVYYGVPVWKKVVEIE PLGVAPTKAKRRVVEREKRGGGG 39 G 

K IAANNLWVTVYYGVPVWKKVVQIE PLGIAPTRARRRVVQREKRGGGG 40 G 

Example 3 Synthesis of DNA Fragments Encoding Chimeric TargetPolypeptides gp120 V3-V3 and gp120 C1-C5-gp41

The nucleotide sequences of the DNA fragments encoding chimeric targetpeptides gp 120 V3-V3 and gp120 C1-C5-gp41 were modified to increasetranslation efficiency without changing the amino acid sequences of theencoded proteins, using the method disclosed in Taiwan patentapplication No. 092126644, which is incorporated herein in its entiretyby reference. The sequence modification allowed the encoded peptides orproteins to be efficiently expressed in E. coli pET plasmid expressionsystem. Table 4 lists the modified sequences of the DNA fragmentsencoding the chimeric target peptides gp120 V3-V3 and gp120 C1-C5-gp41,in which non-italic, capital letters denote restriction enzyme linkersfor EcoR1, Nde1 and Sal1 cutting sites, and italic, capital lettersdenote a XhoI restriction enzyme site.

TABLE 4 Chimeric target SEQ ID peptide Nucleotide sequence No gp120GAATTCCATATGGTCGACatcggttgcacccgtccgagcaacaacacccgtaaaggtatccac 8 V3-V3atgggcccgggtggtgctttctacaccaccggtcagatcatccgtaacatccgtcaggctcactgtggtctgctgggtggttgtacccgtccgaacaacaacacccgtcgtagcatccacatcgaaccggaaggtgctttctacaccaccggtgaaatcatcggtgacatccgtcaggctcactgtggcctgggtCTCGAG gp120GAATTCCATATGGTCGACgttgaaaaactgtgggttaccgtttactacggtgttccggtttggaaa 9C1-C5-gp41aaagttgttaaaatcgaaccgctgggtgttgctccgaccaaatgcaaacgtcgtgttgttcagcgtgaaaagcgtggtggcggtggcggtcaagctcgtgttatcgctgttgaacgttacctgaaagaccagcagctgctgggtatctggggtggtagcggtaaactgatctgctgcaccaccgctgttccgtggaacagcagctggagcaacaaactggaccgtatctggaacaacatgacctggCTCGAG

Table 5 lists SEQ ID NOs. of respective primer pairs used for PCRsynthesis of the DNA fragments encoding chimeric target peptides gp120V3-V3 (SEQ ID NO: 8) and gp120 C1-C5-gp41 (SEQ ID NO: 9).Non-DNA-template PCR reactions were performed by continuously usingforward and reverse primer pairs to PCR synthesize DNA fragments. In thefirst-round PCR, the 3′ end of the first forward primer (F1) had about10-15 bases that were complementary to those in the pairing reversedprimer (R1). The PCR profile was as follows: 5 min at 95° C., 1 min at94° C., 0.5 min at 55° C., 1 min at 72° C. for 20 cycles, and 1 min at72° C. Following the first-round PCR, the 3′ ends of the primer pairs(such as F2 and R2, F3 and R3, F4 and R4, or F5 and R5) had about 10-15bases that were complementary to the previous round PCR product as a DNAtemplate. After the first-round PCR, 0.01-1 μl of the product was usedas the DNA template for the second-round PCR. The second primer pair, F2and R2, were added in a suitable amount together with dNTPs, reagentsand Pfu polymerase, and the second round PCR was performed. Otherprimers were subsequently added in this manner so that the finalextended DNA fragments were synthesized. All the DNA fragmentssynthesized from each primer pair and each round of PCR were analyzedfor the size by gel electrophoresis. The DNA fragment encoding eachindividual chimeric target peptide was synthesized in this manner untilthe final PCR product was extended to the expected size, e.g., 264 bp inthe case of gp120 V3-V3.

The DNA fragments (Table 6) encoding each individual chimeric targetpeptides gp120 C1-C5-gp41 for HIV subtype A, B, C, D, E, F, G, H, J andK were synthesized in a manner similar to the method described aboveusing primer pairs listed in Table 6.

TABLE 5 Chimeric target primer Forward SEQ Reverse SEQ peptide pairsPrimer ID NO. Primer ID NO. gp120 V3-V3 P 1 F1 10 R1 15 P 2 F2 11 R2 16P 3 F3 12 R3 17 P 4 F4 13 R4 18 P 5 F5 14 R5 19 gp120 C1-C5- P 1 F1 20R1 24 gp41 P 2 F2 21 R2 25 P 3 F3 22 R3 26 P 4 F4 23 R4 27 P 5 F4 23 R528 P 6 F4 23 R6 29

Example 4 Construction of Plasmids for Expression of HIV Fusion ProteinsPE(ΔIII)-gp120 V3-V3 and PE(ΔIII)-gp120 C1-C5-gp41

Various chimeric target polypeptides of HIV-1 Env proteins as describedabove were cloned and expressed as PE fusion proteins. Briefly, apolypeptide from Pseudomonas Exotoxin A, i.e., PE(ΔIII), which wasdevoid of cytotoxic domain 111, was fused to respective chimeric targetpolypeptides. Plasmid pPE(ΔIII) was constructed by inserting a DNAfragment encoding the binding domain I and translocation domain II ofPseudomonas exotoxin A into vector pET15a. A DNA sequence coding for acarboxyl terminal peptide that comprises the amino acid sequence KDEL(SEQ ID NO. 30) was ligated to the carboxyl terminal portion of thePE(ΔIII) gene, and generated plasmid pPE(ΔIII)-KDEL3 (referred to aspPE(ΔIII)-K3). The isolated DNA fragments generated by the PCR reactionwere respectively digested by restriction enzymes EcoR I and Xho I, andthen ligated to the EcoR I, Xho I sites of the plasmid pPE(ΔIII)-KDEL3,resulting in chimeric genes that would expressed target peptides asfusion proteins (Hung, C. F. et al (2001) “Cancer Immunotherapy Using aDNA Vaccine Encoding the Translocation Domain of a Bacterial ToxinLinked to a Tumor Antigen” Cancer Research 61:3698-3703).

The plasmid pPE(ΔIII)-HIV gp120 (FIG. 1A) encodes a fusion proteinPE(ΔIII)-HIV gp120 V3-V3 (referred to as PE(ΔIII)-HIV gp120). Theplasmid pPE(ΔIII)-HIV gp120-K3 (FIG. 1B) encodes a fusion proteinPE(ΔIII)-HIV gp120 V3-V3-K3 (referred to as PE(ΔIII)-HIV gp120-K3). Theplasmid pPE(ΔIII)-HIV gp120-41 (FIG. 1C) encodes fusion proteinPE(ΔIII)-HIV gp120 C1-C5-gp41 (referred to as PE(ΔIII)-HIV gp120-41).The plasmid pPE(ΔIII)-HIV gp120-41-K3 (FIG. 1D) encodes the fusionprotein PE(ΔIII)-HIV gp120 C1-C5-gp41-K3 (referred to as PE(ΔIII)-HIVgp120-41-K3). The resulting plasmids were respectively transformed intoE. coli Jam 109 to obtain clones and maintain the clones therein. TheJam 109 strain could stably maintain the plasmid inside bacteria cellswithout protein expression.

TABLE 6 Chimeric Nucleotide target Sequence primer Forward SEQ IDReverse SEQ ID peptide ID NO. pairs Primer NO. Primer NO. A 41 P 1 F1 51R1 56 P 2 F2 52 R2 57 P 3 F3 53 R3 58 P 4 F4 54 R4 59 P 5 F5 55 R5 60 B42 P 1 F1 61 R1 66 P 2 F2 62 R2 67 P 3 F3 63 R3 68 P 4 F4 64 R4 69 P 5F5 65 R5 70 C 43 P 1 F1 71 R1 76 P 2 F2 72 R2 77 P 3 F3 73 R3 78 P 4 F474 R4 79 P 5 F5 75 R5 80 D 44 P 1 F1 81 R1 86 P 2 F2 82 R2 87 P 3 F3 83R3 88 P 4 F4 84 R4 89 P 5 F5 85 R5 90 E 45 P 1 F1 91 R1 96 P 2 F2 92 R297 P 3 F3 93 R3 98 P 4 F4 94 R4 99 P 5 F5 95 R5 100 F 46 P 1 F1 101 R1106 P 2 F2 102 R2 107 P 3 F3 103 R3 108 P 4 F4 104 R4 109 P 5 F5 105 R5110 G 47 P 1 F1 111 R1 116 P 2 F2 112 R2 117 P 3 F3 113 R3 118 P 4 F4114 R4 119 P 5 F5 115 R5 120 H 48 P 1 F1 121 R1 126 P 2 F2 122 R2 127 P3 F3 123 R3 128 P 4 F4 124 R4 129 P 5 F5 125 R5 130 J 49 P 1 F1 131 R1136 P 2 F2 132 R2 137 P 3 F3 133 R3 138 P 4 F4 134 R4 139 P 5 F5 135 R5140 K 50 P 1 F1 141 R1 146 P 2 F2 142 R2 147 P 3 F3 143 R3 148 P 4 F4144 R4 149 P 5 F5 145 R5 150

Example 5 Expression and Analysis of Target Proteins

HIV-1 PE-fusion proteins were expressed in E. coli BL21(DE3) plyscultures containing corresponding expression plasmids. Briefly, 5 ml ofbacterial seeds (A600 of 1.0±0.3 O.D.) were inoculated into 250 ml ofliquid broth (LB) supplemented with 500 μg/ml ampicillin and 50 ml of10% glucose at 37° C. in a rotating incubator shaken at 150 rpm for 2-3hours. Once O.D._(600 nm) reached 0.3±0.1, the bacterial culture wasinduced with isopropylthio-β-D-galactoside (IPTG; Promega, USA) at afinal concentration of 0.1 to 2 mM at 37° C. in a rotating incubatorshaken at 150 rpm for 2 hours for protein expression.

Bacterial cells were pelleted after the protein induction was completed.After freezing and thawing of the pellet, bacterial cells were lysedwith a solution containing in 10 ml: 0.3 mg/ml lysozyme, 1 mMphenylmethylsulfonyl fluoride (PMSF) and 0.06 mg/ml DNase I at roomtemperature for 20 minutes, followed by addition of 1 ml of 10% TritonX-100 and incubation at room temperature for 10 minutes. The lysed cellswere centrifuged at 12,000 g for 10 minutes, and pellets were washed by1 M and 2 M urea solutions. Insoluble inclusion bodies containingrecombinant proteins were collected and dissolved in 8 ml of 8M ureasolution or in an alkaline solution (pH 10 to 12) containing 1 to 3 Murea, and purified using a commercial pET His-Tag purification columnsystem. The protein inclusion bodies dissolved in the urea solution wereloaded onto a 4 ml Ni²⁺-nitrilotriacetic acid (Ni-NTA) resin affinitycolumn, and the bound material eluted by different pH buffers (e.g.pH=8.0, 7.0, 6.5, 6.0, 5.4, and 3.5) containing 1 to 6 M urea with 0.1to 0.3 M NaCl, 5 to 50 mM phosphate buffer, and 5 to 50 mM Tris.

The eluted proteins were analyzed by sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and stained withcoomassie blue. The optical densities of the bands in the gels weremeasured with a densitometer for evaluation of protein quantities. Theconcentration of fusion proteins, e.g., PE(ΔIII)-HIV gp120 C1-C5-gp41-K3(referred to as gp120-41-K3) and PE(ΔIII)-HIV gp120 V3-V3-K3 (referredto as gp120-K3), in the eluted samples were about 0.8 mg/ml.

Example 6 Antibody Assay

Materials and methods. Five PE-HIV envelope peptides were used: (1)PE(ΔIII)-HIV gp120; (2) PE(ΔIII)-HIV gp120-KDEL; (3) PE(ΔIII)-HIVgp120-41; and (4) PE(ΔIII)-HIV gp120-41-KDEL. An oil adjuvant, ISA 206,was used with each of the peptide immunogens in emulsified preparationsfor injection into mice.

Animals. BALB/c mice were purchased form Harlan laboratories and housedin the Laboratory animal Resources Facility of KUMC. All mice were usedin accordance with AAALAC and the KUMC Institutional Animal Care and UseCommittee guidelines.

Immunization of animals with PE-HIV-Env fusion protein vaccines. Four-to six-week old BALB/c mice were divided into 5 groups, with 6 mice pergroup. The animals in groups 1 to 5 received (1) PE(ΔIII)-HIV gp120, (2)PE(ΔIII)-HIV gp120-K3, (3) PE(ΔIII)-HIV gp120-41, (4) PE(ΔIII)-HIVgp120-41-K3, and (5) PBS (control group), in adjuvant, respectively,following the immunization schedule shown in Table 7. Immunized micewere then exsanguinated following deep anesthesia and blood and spleenswere collected for immunological assays.

TABLE 7 Immunization Schedule 1^(st) time 2^(nd) time 3^(rd) time 4^(th)time 5^(th) time No. of *(IM) (IM) (IM) (I/M) **(IP) Group miceImmunogen 0 week 2 weeks 4 weeks 6 weeks 8 weeks 1 6 PE(ΔIII)-HIV gp12050 μg 25 μg 25 μg 25 μg 50 μg Adjuvant 50 μl 50 μl 50 μl 50 μl 0 2 6PE(ΔIII)-HIV gp120-K3 50 μg 25 μg 25 μg 25 μg 50 μg Adjuvant 50 μl 50 μl50 μl 50 μl 0 3 6 PE(ΔIII)-HIV gp120-41 50 μg 25 μg 25 μg 25 μg 50 μgAdjuvant 50 μl 50 μl 50 μl 50 μl 0 4 6 PE(ΔIII)-HIV gp120-41-K3 50 μg 25μg 25 μg 25 μg 50 μg Adjuvant 50 μl 50 μl 50 μl 50 μl 0 5 6 PBS 0 0 0 050 μl Adjuvant 50 μl 50 μl 50 μl 50 μl 0 *IM denotes intramuscularinjection **IP denotes intraperitoneal injection

Serum antibody binding assay. An ELISA test using a commercial kit wasused to determine binding antibody titers. Briefly, two weeks after thelast immunization, mice were anesthetized, sacrificed, and spleens andblood samples were collected. Sera prepared from the blood samples ofthe animals immunized with fusion proteins in groups 1 to 4 and animalsin the placebo group were serially diluted 500×, 2500×, 12500×, and62500×, respectively. Binding antibody titers induced by the fourpeptide groups were analyzed using ELISA kit (BioChain) for detection ofanti-HIV antibodies. Plates were coated with HIV antigen. Themanufacturer's protocol was modified for using mouse sera bysubstituting the anti-human antibody with a goat anti-mouse serumconjugated with Horseradish peroxidase (HRP). Results were expressed asOD absorbance at 450 nm against the blank. FIGS. 2A-2B illustrate thetest results of sera from animals immunized with respective fusionproteins. The absorbance data reflects the amount of antibody titers inserum samples. The result indicates that each fusion protein vaccineafter being injected into animals was able to induce antibodies againstHIV with good titers in the dilution of 1: 2,500 (FIG. 2A) and 12,500(FIG. 2B).

Example 7 Immunized Mouse Sera Neutralize HIV-Attenuated Live VaccineVirus

Neutralization assays were performed using mouse sera against anattenuated live SHIV vaccine virus that was developed in the MMD Lab.The assays were performed in X4 GHOST cells using a plaque reductionassay. X4 GHOST cells were capable of harboring HIV virus plaques afterbeing infected by an HIV-attenuated live vaccine virus. Briefly, serumsamples collected from immunized mice were tested for their contents ofHIV specific neutralizing antibodies. Briefly, quadruplicates of serialtwofold dilutions of sera in RPMI 1640 medium were prepared in 96-wellplates. Twenty plaque-forming units of virus were incubated with thetwofold dilutions of serum samples from each immunized mouse and anormal serum for two hours at 37 C, respectively. The suspensions werethen inoculated onto monolayers of X4 GHOST cells that constitutivelyexpressed the HIV LTR linked to GFP. These cultures were incubated forthree days and then examined by immunofluorescence for determination ofthe numbers of fluorescence spots that represented successful virushits. Neutralization titers of the serum samples were scored as thehighest dilution of the immune serum sample that prevented developmentof 50% of the plaques induced with the control nonimmune serum. Micedeveloped neutralizing antibodies against the live vaccine virus (FIG.3). The highest serum neutralization titers were obtained with sera frommice immunized with PE(ΔIII)-HIV gp120-41-KDEL.

Example 8 Immunized Mouse Eera Neutralize Simian-Human ImmunodeficiencyVirus (SHIV_(KU2))

Immunization of animals. Mice were immunized with higher dosages ofPE-fusion protein vaccines using a similar protocol described above.Briefly, mice were divided into 5 groups (groups 6-10), with 6 mice pergroup, and received PE(ΔIII)-HIV gp120, gp120-K3, PE(ΔIII)-HIV gp120-41,PE(ΔIII)-HIV gp120-41-K3, and PBS (control group), in adjuvant,respectively. The immunization schedule is shown in Table 8.

TABLE 8 Immunization First time Second time Third time No. of *(IM) (IM)**(IP) Group mice Immunogen 0 week 2 weeks 6 weeks 6 6 PE(ΔIII)-HIVgp120100 μg 50 μg 50 μg Adjuvant 100 μl 50 μl 0 7 6 PE(ΔIII)-HIVgp120-K3 100μg 50 μg 50 μg Adjuvant 100 μl 50 μl 0 8 6 PE(ΔIII)-HIVgp120-41 100 μg50 μg 50 μg Adjuvant 100 μl 50 μl 0 9 6 PE(ΔIII)-HIVgp120-41-K3 100 μg50 μg 50 μg Adjuvant 100 μl 50 μl 0 10 6 PBS 100 μl 50 μl 50 μl Adjuvant100 μl 50 μl 0 *IM: intramuscular injection with adjuvant ISA 206. **IP:intraperitoneal injection with adjuvant ISA 206.

TABLE 9 No. of live mice/No. of Neutralization Group Vaccine challengedmice titer 6 PE(ΔIII)-HIV gp120 4/6 1:20 7 PE(ΔIII)-HIV gp120-K3 1/61:20 8 PE(ΔIII)-HIV gp120-41 5/6 1:20 9 PE(ΔIII)-HIV gp120-41-K3 3/61:20-40 10 PBS/adjuvant* 0/6 — *Adjuvant: ISA 206.

Two weeks after the third immunization, blood samples were collectedfrom each group of animals and processed to obtain serum samples forassay of antibody titers. ELISA antibody assays showed similar titers inthis experiment as those in Example 6.

To perform neutralization assays using SHIV_(KU2), mice from each groupwere challenged by pathogenic SHIV_(KU2) vaccine (constructed by Dr.Narayan, University of Kansas Medical Center, U.S. Pat. No. 5,849,994).All four groups of the immunized animals developed neutralizing antibodytiters of approximate 1:20 against SHIV_(KU2). Table 9 shows thesurvival data. PE(III) gp120-41 was the best antigen preparation forinduction of neutralizing antibodies against SHIV_(KU2) since 5 out of 6mice developed these antibodies.

Example 9 HIV Gag24, Nef, Tat, and Rev Fusion Proteins

Materials and methods. Four HIV fusion proteins were tested for theirimmunogenicity: (I) HIV Gag24 fusion protein vaccines, PE(ΔIII)-HIVGag24-K3 and PE(ΔIII)-HIV Gag24-gp120-41-K3; (II) HIV Nef fusion proteinvaccines comprising PE(ΔIII)-HIV Nef-N-K3 and PE(ΔIII)-HIV Nef-C-K3;(III) HIV Tat fusion protein vaccine comprising PE(ΔIII)-HIV tat-K3; and(IV) HIV Rev fusion protein vaccine comprising PE(ΔIII)-HIV Rev-K3. Theabove (I) to (IV) HIV fusion proteins were constructed using similarmethods described in Examples 2 to 4. Briefly, various polypeptidesegments (Table 10) were selected from HIV proteins Gag24, Nef, Tat andRev, respectively.

TABLE 10 Targeted HIV peptide SEQ ID Proteins segmentsAmino acid sequence No. Gag24 Full VDRDELKGIGMTNNPPIPVGEIYKRWIILGLNKIVRM151 length YSPTMTNNPPIPVGEIYRWIILGLNKIVRMYSPT Gag24 Nef Nef- NPTVRQRMDRTEPAAEGVGAVSRDLEKHGAITSSNTA 152 terminusATNADCAWLEAQEEEEVGFPVRPQVPLRPMTYKAAVDISHFLKEKGGLEGLIYSQKRQEILDLWIYHTQGYF PDWQNYTPGPGIRYPLTFGWCFKL Nef- CFLKVPVDPEQVEKANEGDNNCLLHPISQHGMDDPE 153 terminusKEVLMWKFDSRLAFQHIAREKHPEYYKDCLG Tat FullRDELKGIGMEPVDPRLEPWKHPGSQPRTACNNCYC 154 lengthKKCCFHCPVCFISKGLGISYGRKKRRQRRRAPQDSE TatTHQVSLSKQPTSQLRGDPTGPKESKKKVERETETDP NV Rev FullLLAVRIIKTLYQSNPYPKPEGYRRVRRNRRRRWRAR 155 lengthQRQIHSISERILITCLGRPTEPVPLQLPPIERLNINCSES Rev GGTSGTQRVGNP

TABLE 11 Nucleotide Target Sequence primer Forward SEQ ID Reverse SEQ IDpeptide ID NO. pairs Primer NO. Primer NO. Gag24 156 P1 F1 161 R1 165 P2F2 162 R2 166 P3 F3 163 R3 167 P4 F4 164 R4 168 Nef-N 157 P1 F1 169 R1176 terminus P2 F2 170 R2 177 P3 F3 171 R3 178 P4 F4 172 R4 179 P5 F5173 R5 180 P6 F6 174 R6 181 P7 F7 175 R7 182 P8 F7 175 R8 183 P9 F7 175R9 184 Nef-C 158 P1 F1 185 R1 189 terminus P2 F2 186 R2 190 P3 F3 187 R3191 P4 F4 188 R4 192 Tat 159 P1 F1 193 R1 200 P2 F2 194 R2 201 P3 F3 195R3 202 P4 F4 196 R4 203 P5 F5 197 R5 204 P6 F6 198 R6 205 P7 F7 199 R7206 Rev 160 P1 F1 207 R1 213 P2 F2 208 R2 214 P3 F3 209 R3 215 P4 F4 210R4 216 P5 F5 211 R5 217 P6 F6 212 R5 217

The DNA fragments (Table 11) encoding respective polypeptide segmentswere synthesized using primers listed in Table 11 by multi-round PCRsynthesis method as described previously. Gel electrophoresisexperiments were performed to examine the PCR products generated by eachprimer pair in the multiple-round PCR synthesis of DNA fragments, e.g.,410, 412, 414, 416 (FIG. 4). The PCR synthesized DNA fragments werefused to a PE fragment, cloned and expressed, respectively, using asimilar method described in Example 4. The PE fragment is a polypeptidecalled PE (ΔIII) that contains a binding domain and a translocationdomain from Pseudomonas Exotoxin A but lacks a cytotoxic domain. Forexample, the DNA fragment encoding gag24 generated by PCR was digestedby restriction enzymes EcoRI and Xho I to isolate a 225 bp fragment,402, which was ligated to a PE(ΔIII) fragment within an EcoRI and XhoI-digested plasmid pPE(ΔIII)-KDEL3, 401, to generatepPE(ΔIII)-HIV-gag24-K3 (FIG. 4).

A 1.7 Kb gp120-41 fragment, isolated from Sal I and Pst I-digestedpPE(ΔIII)-HIV gp120-41-K3, 502, was fused to gag24 by insertingdownstream, or C-terminal, to the gag24 gene within a Pst I, XhoI-digested plasmid pPE(ΔIII)-HIV gag24-K3, 501, to generate plasmidpPE(AH1)-HIV gag24-gp120-41-K3 (FIG. 5). Plasmid pPE(ΔIII)-HIV nef-C-K3was digested by Sal I and Pst I to isolate a 1.6 Kb fragment, 702,followed by ligation to a nef-N fragment within a Pst I, Xho I-digestedplasmid pPE(ΔIII)-HIV nef-N-KDEL, 701, to generate pPE(ΔIII)-HIVnef-NC-KDEL (FIG. 7).

The DNA fragment encoding Tat or Rev generated by PCR was digested byrestriction enzymes EcoRI and Xho I to isolate fragment, which wasligated to a PE(ΔIII) fragment within an EcoRI and Xho I-digestedplasmid pPE(ΔIII)-KDEL3. By employing DNA recombinant method, plasmidspPE(ΔIII)-HIV Tat-K3 (FIG. 1F), and pPE(ΔIII)-HIV Rev-K3 (FIG. 1E) weregenerated.

The sequence KDEL3 (i.e., K3) is an endoplasmic reticulum (ER) retentionpeptide located at the carboxyl terminal portion of the chimeric fusionprotein. The sequence listing illustrates the nucleotide sequences ofPE(ΔIII)-HIV Gag24-K3, PE(ΔIII)-HIV gag24-gp120-41-K3, PE-(ΔIII)-HIVnef-N-K3, pPE(ΔIII)-HIV nef-C-K3, pPE(ΔIII)-HIV-nef-NC-K3, pPE(III)-HIVrev-K3, pPE(ΔIII)-HIV-tat-K3 as SEQ ID NOs: 236, 238, 240, 242, 244, 246and 248, and the corresponding amino acid sequences as SEQ ID NOs: 237,239, 241, 243, 245, 247, and 249, respectively. For clinicalapplications, any undesired sequence such as oncogen sequences, ifpresent in the bridge between PE(ΔIII) and HIV target peptide (e.g.,between EcoRI and AatII), may be deleted without affecting the HIVtarget antigenic determinants.

Immunization of animals with fusion proteins. Chimeric fusion proteinsas described above were expressed for vaccination. Female mice C57BL/6Jaged 6- to 8-week old were purchased from National Taiwan University(Taipei, Taiwan) and bred in Animal Center of National Taiwan UniversityHospital. Mice were divided into groups and injected three times attwo-week intervals with respective fusion proteins or PBS (controlgroup) in ISA 206 oil adjuvant (Table 12). Two weeks after the lastimmunization, mice were exsanguinated under deep anesthesia and bloodand spleens were collected for immunological assays. An ELISA test usinga commercial kit was used to determine binding antibody titers.

TABLE 12 Immunization Schedule HIV target No. of First time Second timeThird time peptides Vaccine* Mice 0 week 2 weeks 4 weeks PlaceboPBS/Adjuvant 3 100 μl 100 μl 100 μl Gag24 PE(ΔIII)-HIV Gag24-K3 4 100 μg100 μg 100 μg gp120 PE(ΔIII)-HIV gp120-41-K3 4 100 μg 100 μg 100 μgGag24-gp120-41 PE(ΔIII)-HIV Gag24-gp120-41-K3 3 100 μg 100 μg 100 μg NefPE(ΔIII)-HIV Nef-N-K3 4 100 μg 100 μg 100 μg PE(ΔIII)-HIV Nef-C-K3 (50μg each) (50 μg each) (50 μg each) Tat PE(ΔIII)-HIV Tat-K3 4 100 μg 100μg 100 μg Rev PE(ΔIII)-HIV Rev-K3 4 100 μg 100 μg 100 μg *Vaccines wereprepared by mixing fusion proteins or PBS with 50 μl of adjuvant ISA 206before intramuscular (IM) injection.

Serum Antibody Test. Sera from mice were prepared and serially diluted500×, 2500×, 12500×, and 62500× using the method described in Example 5.Antibody titers were measured using indirect ELISA analysis. ELISAplates were prepared and coated with corresponding peptides in Tables 13and 14.

Cytokine release assay. Splenocyte cytokine levels in the medium ofcultured cells were examined by ELISA to measure levels of cytokinesTNF-α, γ-IFN, IL-4, IL-10 and IL-12. Briefly, spleens were asepticallycollected from mice and dissociated to harvest splenocytes. Cells wereresuspended in RPMI, and mononuclear cells were counted in ahemocytomer. Splenocytes were diluted to an optimal density and culturedin 5×10⁶ cells/well in 6-well plates in 5 ml of RPMI. Immunogen inducerpeptides, e.g., Gag24-N and Gag24-C, etc., were respectively added tosplenocytes in triplicate. On the second day after the addition ofimmunogens, supernatants were collected. The amounts of TNF-α, γ-IFN,IL-4, IL-10 and IL-12 produced by splenocyte CD8+ T cells were assayedusing quantitative ELISA assay kits (Invitrogen BioSource) by followingthe manufacturer's protocol with slight modifications.

TABLE 13 Antigen  SEQ ID Length peptide Peptide sequence NO. (a.a.)gp120-41-N1 VEKLWVTVYYGVPVWK 218 16 gp120-41-N2 KVVKIEPLGVAPTKCK 219 16gp120-41-N3 APTKCKRRVVQREKR 220 15 gp120-41-C1 QARVWRYLKDQQLL 221 14gp120-41-C2 GIWGCSGKLICCTTAVP 222 17 gp120-41-C3 AVPWNASSWSNKLDR 223 15

TABLE 14 SEQ ID Length Antigen peptide Peptide sequence NO. (a.a.)HIV-Tat-N PVDPRLEPWKHPGSQPRTAC 224 20 HIV-Tat-C QLRGDPTGPKESKKKVERET 22520 HIV-Tat-M SYGRKKRRQRRRAPQDSETH 226 20 HIV-Rev-N QSNPYPKPEGYRRVRRNRRR227 20 HIV-Rev-C NCSESGGTSGTQRVGNPLEK 228 20 HIV-Nef-n1-NSKLKKGWPTVRQRMDRTE 229 18 HIV-Nef-n1-C TQGYFPDWQNYTPGPGIR 230 18HIV-Nef-c1-N VDPEQVEKANEGDNN 231 15 HIV-Nef-c1-M ISQHGMDDPEKEVLM 232 15HIV-Nef-c1-C QHIAREKHPEYYKDCLGLEK 233 20 HIV-Gag24-N PEFHMVDRDELKGIGMTN234 18 HIV-Gag24-C RMYSPTMTNNPPIPV 235 15

Spleen lymphoid cell proliferation CMI assay. Cell proliferation ELISABrdU (colorimetric) assays for CMI reactions were performed. The stepsfor culturing splenocytes were similar to those used in cytokine releaseassay except that cells were cultured in 96-well plates. Briefly,immunogens or antigen peptides, e.g., Gag24-N, Gag24-C, etc., wererespectively added to cell culture on day-2 to stimulate cellproliferation. ConA (10 μg/ml), as a positive control, was added tostimulate cell2s for one day. Cells were pulse-labeled with BrdU onday-3 at 37° C. for 12-24 hr. Only proliferating cells incorporated BrdUinto their DNA. Cells were fixed with FixDenat solution. The FixDenatsolution also denatured the genomic DNA, exposing the incorporated BrdUto immunodetection. The BrdU label in the DNA was located with aperoxidase-conjugated anti-BrdU antibody (anti-BrdU-POD). The boundanti-BrdU-POD was quantitated with a peroxidase substrate TMB bymeasuring absorbance at OD650 using ELISA plate reader.

Table 15 shows Gag24-specific antibodies titers in immunized mouse Sera.The antibody titer assay indicated that Gag24-N antigenic determinant orepitope peptide was stronger in inducing antibody reactions than theGag24-C epitope peptide. The ability of Gag24-N peptide in inducingantibody titers was, however, weak when it was in the fusion proteinPE(ΔIII)-Gag24-K3. Once the Gag24-N antigenic determinant peptide wasmodified to include polypeptide gp120 and gp41 α-helix to form fusionprotein PE(ΔIII)-Gag24-gp120-41-K3, its ability of inducingGag24-N-specific IgG increased significantly. Thus, the peptide Gag24-Ncould elicit a Th2 cell-dependent, antigenic determinant (orepitope)-specific humoral immune response.

TABLE 15** Antibody IgG IgA IgE Mouse No. Coated Pl* Pl Pl VaccineAntigen #1 #2 #3 #1 #1 #2 #3 #1 #1 #2 #3 #1 PE(ΔIII)- Gag24-N 10 10 10 13 3 3 1 1 1 3 1 Gag24-K3 Gag24-C 3 3 3 1 10 3 3 1 1 3 1 1 PE(ΔIII)-Gag24-N 100 100 100 1 3 3 3 1 3 3 3 1 Gag24- Gag24-C 3 3 10 1 3 3 3 1 11 1 1 gp120-41- K3 *The term “Pl” denotes “placebo,” in which mice wereinjected with PBS/adjuvant. **The data represented here were endpointsof serum semi-log serial dilution. The experiments were repeated inthree mice per immunogen inducer group.

The results from the cell proliferation CMI assay indicated that bothfusion proteins, PE(ΔIII)-Gag24-K3 and PE(ΔIII)-Gag24-gp120-41-K3, afterbeing injected into mice could induce cell-mediated immune response toGag24 antigen (Table 16). The Gag24 antigen, however, had a low efficacyin inducing cell-mediated immune responses in the fusion proteinPE(ΔIII)-Gag24-K3. Once it was modified to fuse with gp120 C1 and C5domains and gp41 α-helix to form PE(ΔIII)-Gag24-gp120 C1-C5-gp41-K3,Gag24 antigen's ability in inducing cell-mediated immune responsessignificantly increased. Thus, PE(ΔIII)-Gag24-gp120 C1-C5-gp4 l-K3 ismuch stronger than PE(ΔIII)-Gag24-K3 in inducing Gag24-specific,cell-mediated responses and cytokine release. As shown in FIG. 6,chimeric polypeptide HIV PE(ΔIII)-gp120 C1-C5-gp41 600 can act as abuilding unit for connecting other HIV antigenic determinant peptide 602and thereby markedly enhance cell-mediated immune responses of theinserted HIV antigenic peptide 602, such as Gag24. Chimeric polypeptideHIV PE(ΔIII)-gp120 C1-C5-gp41 600 includes PE(6,III) 604, HIV gp 120C1-C5-gp41 608, an endoplasmic reticulum retention sequence 610, with abridge or linker 606 in-between. The fusion of an HIV antigenicdeterminant polypeptide 602 with weak CMI responses and HIV gp120C1-C5-gp41 608 results in a chimeric PE-HIV fusion protein 620 thatexhibits enhanced CMI responses specific to the antigenic determinant602.

TABLE 16 CMI assay on immunized mouse splenocytes* Animal Group FusionProtein Immunogen Vaccine Placebo Vaccine Inducer (n = 3) (n = 3)PE(ΔIII)-Gag24-K3 Gag24-N 0.32 0.19 Gag24-C 0.65 0.26 ConA 1.56 0.24PE(ΔIII)-Gag24- Gag24-N 1.31 0.19 gp120-41-K3 Gag24-C 1.25 0.26 ConA1.42 0.20

The data from the cytokine induction test (Table 17) showed that bothvaccines PE(ΔIII)-Gag24-K3 and PE(ΔIII)-Gag24-gp120-41-K3 after beinginjected into mice did not induce detectable IL-4, which indicated thatthey would be better vaccine candidates for HIV. Of the two fusionprotein vaccines, PE(ΔIII)-Gag24-gp120-41-K3 was much more effectivethan PE(ΔIII)-Gag24-K3 in inducing splenocytes to produce large amountsof IL-10 and IL-12. A comparison of Gag24-N and Gag24-C peptides intheir cytokine inducing effects showed that in the PE(ΔIII)-Gag24-K3vaccine group, Gag24-C peptide appeared to have a strongerT-cell-dependent epitope effect than Gag24-N peptide. In thePE(ΔIII)-Gag24-gp120-41-K3 vaccine group, both Gag24-N and Gag24-Cpeptides were capable of inducing cell-mediated immune responses and hadno difference in their effects in inducing cytokine release. The dataindicated that fusion protein PE(ΔIII)-Gag24-K3 had a low efficacy ininducing Gag24-specific cytokine release; however, fusion proteinPE(ΔIII)-Gag24-gp120-41-K3 had a strong effect in elicitingGag24-specific immune responses.

TABLE 17 Cytokines release assay* Cytokine TNF-α γ-IFN IL-4 IL-10 IL-12Immunogen Animal Group Vaccine Inducer Vac** Plac** Vac** Plac** Vac**Plac** Vac** Plac** Vac** Plac** PE(ΔIII)- Gag24-N 8.6 3.4 6.1 5.6 0 0 20 53.6 31.0 Gag24-K3 Gag24-C 12.9 9.6 12.8 12.8 0 0 12 8 36.5 35.6 ConA143.1 53.4 206.4 81.2 9 0 9 4 60.0 36.1 PE(ΔIII)- Gag24-N 70.0 3.4 17.55.6 0 0 136 0 87.6 31.0 Gag24-gp120- Gag24-C 86.6 9.6 24.4 12.8 0 0 1448 67.2 35.6 41-K3 ConA 314 53.4 765.4 81.2 13.8 0 196 4 87.6 36.1 *Thestandard deviation is not shown here. The unit of the concentration isin pg/ml; n = 3 in both vaccine and placebo groups. **The term “Vac”refers to “Vaccine,” and “Plac” refers to “Placebo.”

The data from the immunized mice Sera ELISA test indicated that thefarthest C-terminal portion of Nef-C antigen determinant peptide had thestrongest antibody reaction (Table 18). Based on the antibody-inducingreactions by fusion protein vaccine PE(ΔIII)-Nef-K3, it was concludedthat peptide Nef-C-C was one of the Th2 cell-dependent, HIV antigenicdeterminant sites (Table 18).

TABLE 18** Nef-specific antibody titers in HIV-Nef fusionprotein-immunized mice Antibody IgG IgA IgE Coated Mouse No. VaccineAntigen #1 #2 #3 P1* #1 #2 #3 P1 #1 #2 #3 P1 PE(ΔIII)- Nef-N-N 30 30 301  3 10 10 10  1 1 1 1 Nef-N- Nef-N-C 30 30 30 1 30 30 30 1 1 3 1 1 K3Nef-C-N 30 30 30 1 10 10 10 1 1 1 1 1 PE(ΔIII)- Nef-C-M 30 30 30 1 10 1010 1 1 1 1 1 Nef-C- Nef-C-C  10⁴  10³ 300  1  10² 10 10 1 1 1 1 1 K3*The term “P1” denotes “placebo #1.” Mice in the placebo group wereinjected with PBS/adjuvant. **The data represent the vaccinated mouseserum titers/placebo serum titers of the endpoints of serum semi-logserial dilution. The experiments were repeated in three mice per eachvaccinated group.

TABLE 19 CMI assay on HIV-Nef fusion protein- immunized mousesplenocytes* Animal Groups Immunogen Vaccine Placebo Vaccine Inducer (n= 3) (n = 3) PE(ΔIII)-Nef-N-K3 Nef-N-N 0.7 0.3 and Nef-N-C 0.8 0.2PE(ΔIII)-Nef-C-K3 Nef-C-N 0.5 0.3 Nef-C-M 0.7 0.2 Nef-C-C 1.0 0.3 ConA0.7 0.3

The cell-mediated immune responses in immunized mice indicated that bothPE(ΔIII)-Nef-N-K3 and PE(ΔIII)-Nef-C-K3 had Nef-antigen-specific CMIreactions, and among which the Nef-N-C and Nef-C-C antigenic determinantportions induced stronger CMI responses (Table 19).

TABLE 20* Cytokines release assay from HIV-Nef fusion protein-immunizedmouse splenocytes Cytokine TNF-α γ-IFN IL-4 IL-10 IL-12 Immunogen AnimalGroup Vaccine Inducer Vac*** Plc*** Vac Plc Vac Plc Vac Plc Vac PlcMixture of Nef-N-N 46.8 2.1 38.9 9.2 0 0 PE(ΔIII)- Nef-N-C 42.8 4.9 34.426.7 0 0 HIV-Nef-N-K3 Nef-C-N 65.2 12.7 61.7 30.9 0 0 143 0 55 38 andNef-C-M 47.8 6.9 27.0 17.4 0 0 PE(ΔIII)- Nef-C-C 51.2 2.9 25.1 10.6 0 0HIV-Nef-C-K3 ConA 80.8 14.7 84.6 15.3 0 0 135 0 69 50 BK** 61.2 10.546.3 22.5 *conc. (pg/ml); n = 3 in both vaccine and placebo group. **BKrefers to “Blank,” in which no immunogen inducer was added intosplenocytes. ***Vac refers to “Vaccine,” and Plc refers to “Placebo.”

The data from the cytokine induction test showed that Nef fusionproteins did not induce detectable Nef-specific IL-4, which was anindication that Nef fusion proteins would be better vaccine candidatesagainst HIV. The data also indicated that a vaccine compositioncomprising fusion proteins PE(ΔIII)-HIV-Nef-N-K3 and PE(ΔIII)-HIV Nef-Ccould stimulate splenocytes to produce a higher amount of IL-10 (Table20).

The results from serum antibody assay indicated that the N-terminalportion of HIV-1 Tat protein could induce remarkable antibody responses,while the mid-segment and C-terminal portions were weak in inducingantibody responses. Thus, the N-terminal portion of HIV Tat proteincould elicit Th2 cell-dependent, antigenic determinant-specific humoralimmunity (Table 21).

The results from the cell immune response indicated that PE(ΔIII)-Tat-K3could induce cell mediated immune responses to all Tat protein segments,among which the N-terminus of Tat protein was stronger than mid- andC-terminal segments in inducing cell immune responses (Table 22).

TABLE 21 Antibody titers in HIV-Tat-fusion protein-immunized miceAntibody IgG IgA IgE Coated Mouse No. Vaccine Antigen #1 #2 #3 #4 P1 #1#2 #3 #4 P1 #1 #2 #3 #4 P1 PE Tat-N  10³  10³  10³  10³ 1 10³ 10³ 10³10³ 1 ? ? 10 10 1 (ΔIII)-HIV- Tat-M 10 10 10 10 1 3 3 3 3 1 3 3 3 3 1Tat-K3 Tat-C 10  1  3  3 1 3 3 3 3 1 1 1 1 1 1 *The term “P1” refers to“placebo #1.” The data represent vaccinated mouse serum titers/placeboserum titers of the endpoints of serum semi-log serial dilution.

TABLE 22 CMI assay on HIV-Tat-fusion protein immunized mousesplenocytes* Animal Groups Immunogen Vaccine Placebo Vaccine Inducer (n= 4) (n = 4) PE(ΔIII)-HIV- Tat-N 1.1 0.2 Tat-K3 Tat-M 1.0 0.2 Tat-C 0.50.2 ConA 1.3 0.2

The results from cytokine release assay indicated that HIV Tat fusionprotein was not able to induce a detectable level of Tat-specific IL-4.Its effects in inducing γ-IFN and TNF-α release were not obvious,either. The fusion protein PE-(ΔIII)-Tat-K3, however, was able tostimulate splenocytes to produce Tat-N terminus-specific IL-12. Thus,PE-(ΔIII)-Tat-K3 was still effective in inducing a cell immune responsethat was specific to the N terminal portion of Tat and therefore theN-terminus of Tat could evoke Th1 cell-dependent, antigenicdeterminant-specific cell mediated immune responses in the PE deliverysystem of the present invention (Table 23).

The antibody data from PE(III)-Rev-K3 fusion protein-immunized micemodel indicated that the antibody responses to HIV-Rev antigenicdeterminant peptide was not strong enough to confirm the locus of Th2cell-dependent, antigenic determinant in Rev protein (Table 24).

TABLE 23 Cytokines release assay on HIV-Tat fusion proteinimmunized-mouse splenocytes* Cytokine TNF-α γ-IF2N IL-4 IL-10 IL-12Immunogen Animal Group Vaccine Inducer Vac Plac Vac Plac Vac Plac VacPlac Vac Plac PE(ΔIII)- Tat-N 10 5 0 18  0 1  6  0 41 10 HIV- Tat-M 18 57 4 — — — — — — Tat-K3 Tat-C 32 9 21 17 — — — — — — BK 10 18 2 27 — — —— — — ConA 155 27 243 81 33 0 48 25 55 27 *conc. (pg/ml); n = 3 in bothvaccine (Vac) and placebo (Plac) group.

TABLE 24 Antibody titers in HIV-Rev fusion protein-immunized miceAntibody IgG IgA IgE Mouse No. Vaccine Immunogen #1 #2 #3 #4 P1* #1 #2#3 #4 P1 #1 #2 #3 #4 P1 PE(III)-HIV- HIV-Rev-N 10 10 10 3 1 3 10 3 10 11 1 1 1 1 Rev-K3 HIV-Rev-C 3 3 1 1 1 3 3 3 3 1 1 1 1 1 1 *The term “P1”denotes “placebo #1.” Mice in the placebo group were injected withPBS/adjuvant. The data represent vaccinated mouse serum titers/placeboserum titers of the endpoints of serum semi-log serial dilution.

The data from cell immune responses in Table 25 indicated thatPE(III)-Rev-K3 fusion protein vaccine was not able to induce cell immuneresponse to Rev antigen. The cytokine release inducing test also gavethe similar result. It showed no obvious effects in inducing TNF-α andγ-IFN release (Table 26). Thus, the fusion protein vaccinePE(ΔIII)-Rev-K3 might not be a good component in an HIV vaccine.However, whether Rev is able to elicit a CMI response under thecondition of fusion with gp 120-41 in a PE delivery system remains to beinvestigated. A plasmid pPE(ΔIII)-HIV-Rev-gp120-41-K3 was constructedfor investigation.

TABLE 25 CMI assay on HIV-Rev fusion protein- immunized mouseSplenocytes Animal Groups Immunogen Vaccine Placebo Vaccine Inducer (n =3) (n = 3) PE(ΔIII)-HIV- Rev-N 0.39 0.25 Rev-K3 Rev-C 0.39 0.24 ConA0.40 0.24

TABLE 26 Cytokines release assay on HIV-Rev fusion protein-immunizedmouse Splenocytes* Cytokine TNF-α γ-IFN Animal Group Immunogen VaccinePlacebo Vaccine Placebo Vaccine Inducer (n = 4) (n = 4) (n = 4) (n = 4)PE(ΔIII)-HIV- Rev-N 29 3 6 4 Rev-K3 Rev-C 30 3 7 5 BK 38 4 9 7 ConA 50 616 5 *conc. (pg/ml)

The foregoing description of the exemplary embodiments of the inventionhas been presented only for the purposes of illustration and descriptionand is not intended to be exhaustive or to limit the invention to theprecise forms disclosed. Many modifications and variations are possiblein light of the above teaching.

The embodiments and examples were chosen and described in order toexplain the principles of the invention and their practical applicationso as to enable others skilled in the art to utilize the invention andvarious embodiments and with various modifications as are suited to theparticular use contemplated. Alternative embodiments will becomeapparent to those skilled in the art to which the present inventionpertains without departing from its spirit and scope. Accordingly, thescope of the present invention is defined by the appended claims ratherthan the foregoing description and the exemplary embodiments describedtherein. All references cited and discussed in this specification areincorporated herein by reference in their entireties and to the sameextent as if each reference was individually incorporated by reference.

1. A method for inducing HIV antigen-specific immune responses,comprising: administering to a subject in need thereof with an effectiveamount of a chimeric fusion protein comprising: a) a first polypeptidylregion comprising a Pseudomonas Exotoxin A (PE) binding domain and a PEtranslocation domain, located at the N-terminus of the fusion protein;and b) a second polypeptidyl region with a fusion peptide of HIVgp120-C1-C5-gp41 with the amino acid sequence of SEQ ID NO:
 7. 2. Amethod as claimed in claim 1, wherein the fusion protein furthercomprises an endoplasmic reticulum retention sequence at the C-terminusof the fusion protein.
 3. A method as claimed in claim 2, wherein thefusion protein further comprises an intermediate polypeptidyl regionwith the amino acid sequence of HIV Gag24 between the first and thesecond polypeptidyl regions.
 4. A method as claimed in claim 2, whereinthe endoplasmic reticulum retention sequence comprises the amino acidsequence KDEL.
 5. A method for inducing neutralizing antibodies againstHIV-1, comprising: administering to a subject in need thereof with aneffective amount of a chimeric fusion protein comprising: a) a firstpolypeptidyl region comprising a Pseudomonas Exotoxin A (PE) bindingdomain and a PE translocation domain, located at the N-terminus of thefusion protein; and b) a second polypeptidyl region with a fusionpeptide of HIV gp120-C1-C5-gp41 with the amino acid sequence of SEQ IDNO:
 7. 6. A method as claimed in claim 5, wherein the fusion proteinfurther comprises an endoplasmic reticulum retention sequence at theC-terminus of the fusion protein.
 7. A method as claimed in claim 6,wherein the fusion protein further comprises an intermediatepolypeptidyl region with the amino acid sequence of HIV Gag24 betweenthe first and the second polypeptidyl regions.
 8. A method as claimed inclaim 7, wherein the endoplasmic reticulum retention sequence composesthe amino acid sequence KDEL.
 9. A chimeric fusion protein comprising:a) a first polypeptidyl region comprising a Pseudomonas Exotoxin A (PE)binding domain and a PIE translocation domain, located at the N-terminusof the fusion protein; and b) a second polypeptidyl region with a fusionpeptide of HIV gp120-C1-C5-gp41 with the amino acid sequence of SEQ IDNO:
 7. 10. A fusion protein as claimed in claim 9, further comprising anendoplasmic reticulum retention sequence at the C-terminus of the fusionprotein.
 11. A fusion protein as claimed in claim 10, further comprisingan intermediate polypeptidyl region with the amino acid sequence of HIVGag24 between the first and the second polypeptidyl regions.
 12. Afusion protein as claimed in claim 10, wherein the endoplasmic reticulumretention sequence comprises the amino acid sequence KDEL.